Hybrid and tandem expression of neisserial proteins

ABSTRACT

Two or more Neisserial proteins are joined such that they are translated as a single polypeptide chain. Hybrid proteins are represented by the formula NH 2 -A-[-X-L-] n -B—COOH where X is an amino acid sequence, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1. Proteins where each of the n -X- moieties shares sequence identity to each other -X- moiety, the protein is a ‘tandem protein’.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.10/488,786, filed Feb. 25, 2005, which is the National Stage ofInternational Patent Application of PCT/IB2002/003904, filed Sep. 6,2002, which claims the benefit of United Kingdom Patent ApplicationSerial No. 0121591.2, filed Sep. 6, 2001, each of which are herebyincorporated by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002100610SeqList.txt,date recorded: Feb. 3, 2012, size: 5499 KB).

TECHNICAL FIELD

This invention is in the field of protein expression. In particular, itrelates to the expression of proteins from Neisseria (e.g. N.gonorrhoeae or, preferably, N. meningitidis).

BACKGROUND ART

References 1 and 2 disclose alternative and improved approaches for theexpression of the Neisserial proteins disclosed in references 3 to 6.One such method is to produce ‘hybrid’ proteins in which two or moreNeisserial proteins are expressed as a single polypeptide chain. Thisapproach offers two advantages. First, a protein that may be unstable orpoorly expressed on its own can be assisted by adding a suitable hybridpartner that overcomes the problem. Second, commercial manufacture issimplified as only one expression and purification need be employed inorder to produce two separately-useful proteins.

It is an object of the present invention to provide further alternativeand improved approaches for the expression of Neisserial proteins.

DISCLOSURE OF THE INVENTION

Hybrid Proteins

Thus the invention provides a method for the simultaneous expression oftwo or more (e.g. 3, 4, 5, 6 or more) Neisserial proteins, in which saidtwo or more proteins are joined such that they are translated as asingle polypeptide chain. In general, the hybrid proteins of theinvention can be represented by the formula: NH₂-A-[-X-L-]_(n)-B—COOH

wherein X is an amino acid sequence, L is an optional linker amino acidsequence, A is an optional N-terminal amino acid sequence, B is anoptional C-terminal amino acid sequence, and n is an integer greaterthan 1.

The value of n is between 2 and x, and the value of x is typically 3, 4,5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; it is more preferably 2or 3; most preferably, n=2.

The -X- Moieties

There are two main groups of hybrid proteins according to the invention.These two groups are not mutually exclusive.

In the first group, each -X- moiety is:

-   -   (a) an orf1, orf4, orf25, orf40, orf46.1, orf83, NMB1343, 230,        233, 287, 292, 594, 687, 736, 741, 907, 919, 936, 953, 961 or        983 amino acid sequence;    -   (b) an amino acid sequence having sequence identity to an amino        acid sequence from (a); or    -   (c) an amino acid sequence comprising a fragment of an amino        acid sequence from (a).

A preferred subset of (a) is: orf46.1, 230, 287, 741, 919, 936, 953, 961and 983. A more preferred subset of (a) is: orf46.1, 287, 741 and 961.FIG. 3 shows preferred hybrid proteins.

In the second group, the hybrid protein comprises a first -X- moiety(-X_(a)-) and a second -X- moiety (-X_(b)-). The -X_(a)- moiety has oneof the following amino acid sequences:

-   -   (d) the 446 even SEQ IDs (i.e. 2, 4, 6, . . . , 890, 892)        disclosed in reference 3.    -   (e) the 45 even SEQ IDs (i.e. 2, 4, 6, . . . , 88, 90) disclosed        in reference 4;    -   (f) the 1674 even SEQ IDs 2-3020, even SEQ IDs 3040-3114, and        all SEQ IDs 3115-3241, disclosed in reference 5;    -   (g) the 2160 amino acid sequences NMB0001 to NMB2160 from        reference 7; or    -   (h) an amino acid sequence disclosed in reference 1 or reference        2.

The -X_(b)- moiety is related to -X_(a)- such that: (i) -X_(b)- hassequence identity to -X_(a)-, and/or (j) -X_(b)- comprises a fragment of-X_(a)-.

Examples of this second type of hybrid protein include proteins in whichtwo or more -X- moieties are identical, or in which they are variants ofthe same protein e.g. two polymorphic forms of the same protein may beexpressed as -X_(a)-X_(b)-, and three polymorphic forms may be expressedas -X_(a)-X_(b)-X_(c)- etc.

The -X_(a)- and -X_(b)- moieties may be in either order from N-terminusto C-terminus.

The -X_(a)- moiety is preferably an orf1, orf4, orf25, orf40, orf46.1,orf83, NMB1343, 230, 233, 287, 292, 594, 687, 736, 741, 907, 919, 936,953, 961 or 983 amino acid sequence. The -X_(a)- moiety is morepreferably an orf46.1, 230, 287, 741, 919, 936, 953, 961 or 983 aminoacid sequence. The -X_(a)- moiety is most preferably an orf46.1, 287,741 or 961 amino acid sequence.

In proteins where each of the n -X- moieties shares sequence identity toeach other -X- moiety, the protein is referred to as a ‘tandem protein’.Tandem proteins in which n=2 are preferred.

The degree of ‘sequence identity’ referred to in (b) and (i) ispreferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more,up to 100%). This includes mutants, homologs, orthologs, allelicvariants etc. [e.g. see ref. 8]. Identity is preferably determined bythe Smith-Waterman homology search algorithm as implemented in theMPSRCH program (Oxford Molecular), using an affine gap search withparameters gap open penalty=12 and gap extension penalty=1. Typically,50% identity or more between two proteins is considered as an indicationof functional equivalence.

The ‘fragment’ referred to in (c) and (j) should consist of least mconsecutive amino acids from an amino acid sequence from (a), (d), (e),(f), (g) or (h) and, depending on the particular sequence, m is 7 ormore (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200 or more). Preferably the fragment comprises an epitopefrom an amino acid sequence from (a), (d), (e), (f), (g) or (h).Preferred fragments are those disclosed in references 9 and 10.

Preferred (c) and (j) fragments are C- and/or N-terminal truncations(e.g. 41-287, 42-287 etc.).

Preferred (b), (c), (i) and (j) sequences omit poly-glycine sequences.This has been found to aid expression [ref. 2]. Poly-glycine sequencescan be represented as (Gly)_(g), where g≧3 (e.g. 4, 5, 6, 7, 8, 9 ormore). If a -X- moiety includes a poly-glycine sequence in its wild-typeform, it is preferred to omit this sequence in the hybrid proteins ofthe invention. This may be by disrupting or removing the (Gly)_(g)—bydeletion (e.g. CGGGGS→CGGGS, CGGS, CGS or CS), by substitution (e.g.CGGGGS→CGXGGS, CGXXGS, CGXGXS etc.), and/or by insertion (e.g.CGGGGS→CGGXGGS, CGXGGGS, etc.). Deletion of (Gly)_(g) is preferred, anddeletion of the N-terminus portion of a protein up to and including thepoly-glycine sequence (e.g. deletion of residues 1-32 in SEQ ID 1) isreferred to herein as ‘ΔG’. Poly-glycine omission is particularly usefulfor proteins 287, 741, 983 and Tbp2 (ΔG287, ΔG741, ΔG983 andΔGTbp2—references 1 & 2).

Preferred (c) and (j) fragments omit complete protein domains. This isparticularly useful for protein 961, 287, and ORF46. Once a protein hasbeen notional divided into domains, (c) and (j) fragments can omit oneor more of these domains (e.g. 287B, 287C, 287BC, ORF46₁₋₄₃₃,ORF46₄₃₄₋₆₀₈, 961c—reference 2; FIGS. 4 and 5 herein).

287 protein has been notionally split into three domains, referred to asA, B & C (see FIG. 5 of reference 2). Domain B aligns with IgAproteases, domain C aligns with transferrin-binding proteins, and domainA shows no strong alignment with database sequences. An alignment ofpolymorphic forms of 287 is disclosed in reference 8.

ORF46 has been notionally split into two domains—a first domain (aminoacids 1-433; ORF46.1) which is well-conserved between species andserogroups, and a second domain (amino acids 434-608) which is notwell-conserved. The second domain is preferably deleted, leavingORF46.1. An alignment of polymorphic forms of ORF46 is disclosed inreference 8.

961 protein has been notionally split into several domains (FIG. 4).

If a -X- moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid proteins of the invention.Where the leader peptide is omitted, this is a preferred example of anamino acid sequence within (c) and (j). In one embodiment, the leaderpeptides will be deleted except for that of the -X- moiety located atthe N-terminus of the hybrid protein i.e. the leader peptide of X₁ willbe retained, but the leader peptides of X₂ . . . X_(n) will be omitted.This is equivalent to deleting all leader peptides and using the leaderpeptide of X₁ as moiety -A-.

When n=2, preferred pairs of -X- moieties are: ΔG287 and 230; ΔG287 and936; ΔG287 and 741; 961c and 287; 961c and 230; 961c and 936; 961cL and287; 961cL and 230; 961cL and 936; ORF46.1 and 936; ORF46.1 and 230; 230and 961; 230 and 741; 936 and 961; 936 and 741. When n=2, preferredpairs of -X- moieties for tandem proteins are: ΔG741 and 741; ΔG287 and287. More specifically, the following combinations of X₁ and X₂ arepreferred when n=2:

X₁ X₂ ΔG287 230 ΔG287 936 ΔG287 741 ΔG287 961 ΔG287 ORF46.1 ΔG287 919ΔG287 953 961c 287 961c 230 961c 936 961c 741 961c 983 961c ΔG983 961cORF46.1 961 ORF46.1 961cL 287 961cL 230 961cL 936 ORF46.1 936 ORF46.1230 ORF46.1 741 ORF46.1 ΔG741 ORF46.1 983 ORF46.1 ΔG983 230 961 230 741230 ΔG741 936 961 936 741 936 ΔG741 ΔG741 741 ORF46.1 983 ΔG741 ORF46.1ΔG741 983 ΔG741 961 ΔG741 961c ΔG983 ORF46.1 ΔG983 961 ΔG983 961c 230ΔG287 936 ΔG287 741 ΔG287 961 ΔG287 ORF46.1 ΔG287 919 ΔG287 953 ΔG287287 961c 230 961c 936 961c 741 961c 983 961c ΔG983 961c ORF46.1 961cORF46.1 961 287 961cL 230 961cL 936 961cL 936 ORF46.1 230 ORF46.1 741ORF46.1 ΔG741 ORF46.1 983 ORF46.1 ΔG983 ORF46.1 961 230 741 230 ΔG741230 961 936 741 936 ΔG741 936 ΔG287 287 983 ORF46.1 ORF46.1 ΔG741 983ΔG741 961 ΔG741 961c ΔG741 ORF46.1 ΔG983 961 ΔG983 961c ΔG983

Where 287 is used in full-length form, it is preferably at theC-terminal end of a hybrid protein; if it is to be used at theN-terminus, if is preferred to use a ΔG form of 287. Similarly, Where741 is used in full-length form, it is preferably at the C-terminal endof a hybrid protein; if it is to be used at the N-terminus, if ispreferred to use a ΔG form of 741.

The -L- Moieties

For each n instances of [-X-L-], linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc.

Linker amino acid sequence(s) -L- will typically be short (e.g. 20 orfewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1). Examples include short peptide sequences whichfacilitate cloning, poly-glycine linkers (i.e. Gly_(n) where n=2, 3, 4,5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His_(n) where n=3,4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acidsequences will be apparent to those skilled in the art. A useful linkeris GSGGGG (SEQ ID 27), with the Gly-Ser dipeptide being formed from aBamHI restriction site, thus aiding cloning and manipulation, and theGly₄ tetrapeptide being a typical poly-glycine linker.

If X_(n+1) is a ΔG protein and L_(n) is a glycine linker, this may beequivalent to X_(n+1) not being a ΔG protein and L_(n) being absent.

The -A- Moiety

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leadersequences to direct protein trafficking, or short peptide sequenceswhich facilitate cloning or purification (e.g. histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- may be a methionineresidue.

The -B- Moiety

-B- is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences whichenhance protein stability. Other suitable C-terminal amino acidsequences will be apparent to those skilled in the art.

Polymorphic Forms of Proteins

The invention can use amino acid sequences from any strains of N.meningitidis. References to a particular protein (e.g. ‘287’, or‘ORF46.1’) therefore include that protein from any strain. Sequencevariations between strains are included within (b), (c), (i) and (j).

Reference sequences from N. meningitidis serogroup B include:

Protein Reference orf1 Ref. 3, SEQ ID 650 orf25 Ref. 3, SEQ ID 684 orf46Ref. 6, SEQ ID 1049 NMB1343 Ref. 7, NMB1343 233 Ref. 5, SEQ ID 860 292Ref. 5, SEQ ID 1220 687 Ref. 5, SEQ ID 2282 741 Ref. 5, SEQ ID 2536 919Ref. 5, SEQ ID 3070 953 Ref. 5, SEQ ID 2918 983 Ref. 7, NMB1969 orf4Ref. 3, SEQ ID 218 orf40 Ref. 4, SEQ ID 4 orf83 Ref. 3, SEQ ID 314 230Ref. 5, SEQ ID 830 287 Ref. 5, SEQ ID 3104 594 Ref. 5, SEQ ID 1862 736Ref. 5, SEQ ID 2506 907 Ref. 5, SEQ ID 2732 936 Ref. 5, SEQ ID 2884 961Ref. 5, SEQ ID 940

Reference 8 discloses polymorphic forms of proteins ORF4, ORF40, ORF46,225, 235, 287, 519, 726, 919 and 953. Polymorphic forms of 961 aredisclosed in references 11 & 12. Any of these polymorphic forms may beused in accordance with the present invention.

The sequence listing herein includes polymorphic forms of proteins 741(SEQ IDs 1-22) and NMB1343 (SEQ IDs 23-24) which have been identified.

Serogroups and Strains

Preferred proteins of the invention comprise -X- moieties having anamino acid sequence found in N. meningitidis serogroup B. Within asingle protein of the invention, individual -X- moieties may be from oneor more strains. Where n=2, for instance, X₂ may be from the same strainas X₁ or from a different strain. Where n=3, the strains might be (i)X₁=X₂=X₃ (ii) X₁=X₂≠X₃ (iii) X₁≠X₂=X₃ (iv) X₁≠X₂≠X₃ or (v) X₁=X₃≠X₂,etc.

Within serogroup B, preferred -X- moieties are from strains 2996, MC58,95N477, or 394/98. Strain 95N477 is sometimes referred to herein as‘ET37’, this being its electrophoretic type. Strain 394/98 is sometimesreferred to herein as ‘nz’, as it is a New Zealand strain.

Where a form of 287 is used, this is preferably from strain 2996 or fromstrain 394/98.

Where a form of 741 is used, this is preferably from serogroup B strainsMC58, 2996, 394/98, or 95N477, or from serogroup C strain 90/18311.

Where a form of 961 is used, this is preferably from strain 2996.

Strains are indicated as a subscript e.g. 741_(MC58) is protein 741 fromstrain MC58. Unless otherwise stated, proteins mentioned herein (e.g.with no subscript) are from N. meningitidis strain 2996, which can betaken as a ‘reference’ strain. It will be appreciated, however, that theinvention is not in general limited by strain. As mentioned above,general references to a protein (e.g. ‘287’, ‘919’ etc.) may be taken toinclude that protein from any strain. This will typically have sequenceidentity to 2996 of 90% or more (eg. 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more).

Domain-Based Expression of Protein 961

References 1 and 2 disclose how a protein can be notionally divided intodomains and how the protein can be manipulated based on these domains.The present invention extends the application of this approach toprotein 961 (also known as ‘NadA’ [11,12]).

In N. meningitidis serogroup B strain 2996, NadA has 405 amino acids.This protein has notionally been divided into the following nine domains(FIG. 4):

Domain name Amino acids 961-1 ‘L’  1-23 961-2  24-87 961-3  88-143 961-4144-180 961-5 181-268 961-6 269-286 961-7 287-330 961-8 331-350 961-9351-405

This information can be used to locate the same domains in other formsof 961.

These domains have been deleted from 961 in strain 2996 in various ways(FIG. 5). Preferred fragments of 961 omit one or more of these ninedomains e.g. the following:

-   -   961-2 to 961-5 (‘961a’)    -   961-6 to 961-9 (‘961b’)    -   961-1 to 961-8 (‘961cL’)    -   961-2 to 961-8 (‘961c’)    -   961-2 to 961-6 and amino acids 287-325 from domain 961-7        (‘961d’)    -   961-2 to 961-8 and amino acids 351-383 from domain 961-9        (‘961Δ1’)    -   961-1 to 961-8 and amino acids 351-383 from domain 961-9        (‘961Δ1L’)    -   961-1 to 961-7 and amino acids 331-343 from domain 961-8        (‘961cL-Δaro’)    -   961-1 to 961-6 and amino acids 287-315 from domain 961-7        (‘961cL-Δcc’)    -   961-1 to 961-5 (‘961aL’)    -   961-1 to 961-4 (‘961aL-Δ1’)    -   961-1 to 961-3 (‘961aL-Δ2’)    -   961-1 to 961-2 (‘961aL-Δ3’)

These thirteen fragments (and sub-fragments thereof missing 1, 2, 3, 4or 5 amino acids at either or both ends) are preferred (c) and (j)fragments, but they may also be expressed in their own right i.e. not inthe form of a hybrid protein of the invention. Thus the inventionprovides a protein comprising one of these fragments, providing that theprotein is not full-length 961 and is not a protein specificallydisclosed in reference 1 or 2. This protein may be a fusion protein(e.g. a GST-fusion or a His-tag fusion).

Sequences

The invention also provides a protein having an amino acid sequence fromSEQ IDs 1 to 24. It also provides proteins and nucleic acid havingsequence identity to these. As described above, the degree of ‘sequenceidentity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%,99% or more).

The invention also provides nucleic acid encoding such proteins.

Furthermore, the invention provides nucleic acid which can hybridise tothis nucleic acid, preferably under “high stringency” conditions (eg.65° C. in a 0.1×SSC, 0.5% SDS solution).

The invention also provides nucleic acid encoding proteins according tothe invention.

It should also be appreciated that the invention provides nucleic acidcomprising sequences complementary to those described above (eg. forantisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared inmany ways (eg. by chemical synthesis, from genomic or cDNA libraries,from the organism itself etc.) and can take various forms (eg. singlestranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and alsotheir analogues, such as those containing modified backbones, and alsopeptide nucleic acids (PNA) etc.

Mixtures

The invention also provides a composition comprising two or more (i.e.2, 3, 4, 5, 6 or 7) of the following proteins:

-   -   (1) 287    -   (2) 741    -   (3) ORF46.1    -   (4) 961    -   (5) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=287, X₂=953    -   (6) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=287, X₂=919    -   (7) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=287, X₂=961

The mixture may include one or both of the following proteins, either incombination with two or more of (1) to (7), or in combination with onlyone of (1) to (7):

-   -   (8) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=287, X₂=741    -   (9) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=936, X₂=741

Where proteins 287 and 741 are included in the mixture (i.e. in protein1, 2, 5, 6, 7 or 8), they may be in the ‘ΔG’ form. Where protein 961 isincluded, it is preferably in the form of ‘961c’ in which the N-terminusleader and C-terminus membrane anchor are absent [e.g. see refs. 1, 2 &11].

A preferred mixture comprises the following three proteins:

-   -   (1) 961c, preferably 961c₂₉₉₆ (e.g. SEQ ID 31 herein);    -   (2) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n is 2, -X₁- is ΔG287        (preferably ΔG287_(NZ)), -X₂- is 953 (preferably 953₂₉₉₆)        lacking its leader peptide, -L₁- is GSGGGG, and -A- comprises a        N-terminus methionine (e.g. -A- is M or MA) (e.g. SEQ IDs 28 &        29 herein); and    -   (3) NH₂-A-[-X-L-]_(n)-B—COOH, wherein n=2, X₁=936 (preferably        936₂₉₉₆), X₂=ΔG741 (preferably ΔG741_(MC58)), L₁=GSGGGG (e.g.        SEQ ID 30 herein).

The mixtures may also comprise N. meningitidis outer membrane vesicles.

Heterologous Host

Whilst expression of the proteins of the invention may take place inNeisseria, the present invention preferably utilises a heterologoushost. The heterologous host may be prokaryotic (e.g. a bacterium) oreukaryotic. It is preferably E. coli, but other suitable hosts includeBacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonellatyphimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g.M. tuberculosis), yeast etc.

Vectors Etc.

The invention provides (a) nucleic acid encoding the proteins describedabove (b) vectors comprising these nucleic acid sequences (c) host cellscontaining said vectors (d) compositions comprising the proteins ornucleic acids of the invention, which may be suitable as immunogeniccompositions (e.g. vaccines) or as diagnostic reagents (e) thesecompositions for use as medicaments (e.g. as vaccines) or as diagnosticreagents (f) the use of these compositions in the manufacture of (1) amedicament for treating or preventing infection due to Neisserialbacteria (2) a diagnostic reagent for detecting the presence ofNeisserial bacteria or of antibodies raised against Neisseria bacteria,and/or (3) a reagent which can raise antibodies against Neisseriabacteria and (g) a method of treating a patient, comprisingadministering to the patient a therapeutically effective amount of thesecompositions.

Implementing the invention will typically involve the basic steps of:obtaining a first nucleic acid encoding a first protein; obtaining asecond nucleic acid encoding a second protein; and ligating the firstand second nucleic acids. The resulting nucleic acid may be insertedinto an expression vector, or may already be part of an expressionvector.

To improve solubility, purification of hybrid proteins may involve therefolding techniques disclosed herein.

Immunogenic Compositions and Medicaments

The compositions of the invention are preferably immunogeniccomposition, and are more preferably vaccine compositions. The pH of thecomposition is preferably between 6 and 7. The pH may be maintained bythe use of a buffer. The composition may be sterile.

Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat infection), but willtypically be prophylactic.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a composition of the invention inthe manufacture of a medicament for raising an immune response in amammal. The medicament is preferably a vaccine.

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of acomposition of the invention. The immune response is preferablyprotective. The method may raise a booster response.

The mammal is preferably a human Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant); wherethe vaccine is for prophylactic use, the human is preferably an adult. Avaccine intended for children may also be administered to adults e.g. toassess safety, dosage, immunogenicity, etc.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by a Neisseria (e.g. meningitis,septicaemia, gonorrhoea etc.). The prevention and/or treatment ofbacterial meningitis is preferred.

Further Components of the Composition

The composition of the invention will typically, in addition to thecomponents mentioned above, comprise one or more ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,trehalose (WO00/56365) and lipid aggregates (such as oil droplets orliposomes). Such carriers are well known to those of ordinary skill inthe art. The vaccines may also contain diluents, such as water, saline,glycerol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent. A thorough discussion of pharmaceutically acceptable excipientsis available in Remington's Pharmaceutical Sciences.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen, as well as any other of the above-mentionedcomponents, as needed. By ‘immunologically effective amount’, it ismeant that the administration of that amount to an individual, either ina single dose or as part of a series, is effective for treatment orprevention. This amount varies depending upon the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.Dosage treatment may be a single dose schedule or a multiple doseschedule (e.g. including booster doses). The vaccine may be administeredin conjunction with other immunoregulatory agents.

The vaccine may be administered in conjunction with otherimmunoregulatory agents.

The composition may include other adjuvants in addition to (or in placeof) the aluminium salt. Preferred adjuvants to enhance effectiveness ofthe composition include, but are not limited to: (1) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59™ (WO90/14837; Chapter 10 inref. 13), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing MTP-PE) formulated into submicron particles usinga microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g.WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e ggamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454; (6)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7)oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622;Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3,849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al.,J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186,1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344;Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J.Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996,167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873;Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J.Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157,4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J.Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160,5898-5906; International patent applications WO96/02555, WO98/16247,WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e.containing at least one CG dinucleotide, with 5-methylcytosineoptionally being used in place of cytosine; (8) a polyoxyethylene etheror a polyoxyethylene ester e.g. WO99/52549; (9) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (e.g.WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol (e.g. WO01/21152); (10) an immunostimulatory oligonucleotide(e.g. a CpG oligonucleotide) and a saponin e.g. WO00/62800; (11) animmunostimulant and a particle of metal salt e.g. WO00/23105; (12) asaponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin(e.g. QS21)+3dMPL+IL-12 (optionally +a sterol) e.g. WO98/57659; (14)other substances that act as immunostimulating agents to enhance theefficacy of the composition.

Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-O-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

Further Antigens

Further antigens which can be included in the composition of theinvention include:

-   -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 14, 15, 16, 17        etc.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 18 from        serogroup C [see also ref. 19] or the oligosaccharides of ref.        20.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        21, 22, 23].    -   a protein antigen from Helicobacter pylori such as CagA [e.g.        24], VacA [e.g. 24], NAP [e.g. 25], HopX [e.g. 26], HopY [e.g.        26] and/or urease.    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 27, 28].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 28, 29].    -   an antigen from hepatitis C virus [e.g. 30].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3[ e.g. refs. 31 & 32].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 33] e.g. the CRM₁₉₇ mutant [e.g. 34].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 33].    -   a saccharide antigen from Haemophilus influenzae B [e.g. 19].    -   an antigen from N. gonorrhoeae [e.g. 3, 4, 5].    -   an antigen from Chlamydia pneumoniae [e.g. 35, 36, 37, 38, 39,        40, 41].    -   an antigen from Chlamydia trachomatis [e.g. 42].    -   an antigen from Porphyromonas gingivalis [e.g. 43].    -   polio antigen(s) [e.g. 44, 45] such as IPV or OPV.    -   rabies antigen(s) [e.g. 46] such as lyophilised inactivated        virus [e.g. 47, RabAvert™]    -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11        of ref. 33].    -   influenza antigen(s) [e.g. chapter 19 of ref. 33], such as the        haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 48].    -   a protein antigen from Streptococcus agalactiae (group B        streptococcus) [e.g. 49, 50].    -   a saccharide antigen from Streptococcus agalactiae    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 50, 51, 52].    -   an antigen from Staphylococcus aureus [e.g. 53].

The composition may comprise one or more of these further antigens.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity [e.g.refs. 54 to 63]. Preferred carrier proteins are bacterial toxins ortoxoids, such as diphtheria or tetanus toxoids. The CRM₁₉₇ diphtheriatoxoid is particularly preferred. Other suitable carrier proteinsinclude the N. meningitidis outer membrane protein [e.g. ref. 64],synthetic peptides [e.g. 65, 66], heat shock proteins [e.g. 67],pertussis proteins [e.g. 68, 69], protein D from H. influenzae [e.g.70], toxin A or B from C. difficile [e.g. 71], etc. Where a mixturecomprises capsular saccharides from both serogroups A and C, it ispreferred that the ratio (w/w) of MenA saccharide:MenC saccharide isgreater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Saccharidesfrom different serogroups of N. meningitidis may be conjugated to thesame or different carrier proteins.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[32]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

Antigens are preferably mixed with (and more preferably adsorbed to) analuminium salt (e.g. phosphate, hydroxide, hydroxyphosphate,oxyhydroxide, orthophosphate, sulphate). The salt may take any suitableform (e.g. gel, crystalline, amorphous etc.).

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 72to 80]. Protein components of the compositions of the invention may thusbe replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein.

DEFINITIONS

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of twenty-three sequences for protein 741.These are SEQ IDs 1 to 22 plus the sequence from MC58.

FIG. 2 shows an alignment of the NMB1343 sequence from gonococcus (top;SEQ ID 25) and meningococcus (bottom; SEQ ID 26).

FIG. 3 shows hybrid and tandem proteins of the invention.

FIG. 4 shows 9 domains within 961₂₉₉₆, and

FIG. 5 shows how these have been manipulated.

MODES FOR CARRYING OUT THE INVENTION

Hybrid Proteins—X₁=ΔG287

In addition to those disclosed in references 1 & 2, seven hybridproteins with ΔG287 from strain 2996 at the N-terminus were constructed.Eight 287 tandem proteins were also made (see below).

# n X₁ L₁ X₂ L₂ 1 2 ΔG287 — 230 (His)₆ 2 2 — 936 (His)₆ 3 2 — 741_(MC58)(His)₆ 4 2 — 741_(ET37) (His)₆ 5 2 — 741_(90/18311) (His)₆ 6 2 —741_(95N477) (His)₆ 7 2 ΔG287_(nz) — 741_(MC58) (His)₆

These proteins were adjuvanted with either Freund's complete adjuvant(FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera weretested against various Neisserial strains using the bactericidal assay.Titres using protein #3 were as follows:

Strain ^((serogroup)) 2996 ^((B)) MC58 ^((B)) NGH38 ^((B)) 394/98 ^((B))44/76 ^((B)) F6124 ^((A)) Al hydroxide 8192 32768 8192 >2048 16384 8192FCA 16384 262144 8192 >2048 >32768 8192

In further experiments using protein #3 adjuvanted with aluminiumhydroxide, anti-287 and anti-741 ELISA titres each exceeded 984150 andBCA titres were as follows:

394/ 44/ 2996 ^((B)) MC58 ^((B)) NGH38 ^((B)) 98 ^((B)) 76 ^((B)) F6124^((A)) BZ133 ^((C)) 8000 65000 4000 4000 32000 8000 16000

Results obtained after immunisation with proteins disclosed in refs. 1 &2, tested against the homologous strain, were as follows:

Bactericidal titre ELISA n X₁ L₁ X₂ L₂ FCA Alum FCA Alum 2ΔG287_(394/98) — 961 (His)₆ — 32768 — >109350 919 32768 4096 4718 3678953 >32768 >16384 1900 6936 741 16384 2048 232 862 2 ΔG287₂₉₉₆ — 961(His)₆ 65536 32768 108627 >109350 919 128000 32000 11851 2581 953 65536— 3834 — 741 16384 8192 315 4645

Hybrid Proteins—X₁=961c or 961cL

In addition to those disclosed in references 1 & 2, eight hybridproteins with either 961c or 961cL (i.e. 961c+leader peptide) at theN-terminus were constructed:

# n X₁ L₁ X₂ L₂ 1 2 961c — 287 — 2 2 — 287 (His)₆ 3 2 — 230 (His)₆ 4 2 —936 (His)₆ 5 2 961cL — 287 — 6 2 — 287 (His)₆ 7 2 — 230 (His)₆ 8 2 — 936(His)₆

These proteins were adjuvanted with either Freund's complete adjuvant(FCA) or 3.3 mg/ml alum and used to immunise mice. The resulting serawere tested against various Neisserial strains using the bactericidalassay. Titres using protein #8 were as follows:

Strain^((serogroup)) 2996^((B)) MC58^((B)) 394/98^((B)) 44/76^((B))F6124^((A)) Al hydroxide 8192 8192 512 1024 <16 FCA 6553616384 >2048 >2048 8192

Titres obtained after immunisation with 961c-741[ refs. 1 & 2] were asfollows:

Strain ^((serogroup)) 2996 ^((B)) MC58 ^((B)) 394/98 ^((B)) 44/76 ^((B))F6124 ^((A)) BZ133 ^((C)) Al hydroxide 65536 32768 4096 >3276816384 >2048 FCA >16384 262144 4096 >16384 — >2048

These results could be improved by mixing 961c-741 with ORF46.1 or withΔG287-919.

Results obtained after immunisation with proteins disclosed in refs. 1 &2, tested against the homologous strain, were as follows:

Bactericidal titre ELISA n X₁ L₁ X₂ L₂ FCA Alum FCA Alum 2 961c —ORF46.1 (His)₆ 32768 1024 >109350 >109350 741 >163848192 >109350 >109350 936 >32768 8192 >109350 >109350

Hybrid Proteins—X₁=ORF46.1

In addition to those disclosed in references 1 & 2, two hybrid proteinswith ORF46.1 at the N-terminus were constructed:

# n X₁ L₁ X₂ L₂ 1 2 ORF46.1 — 936 (His)₆ 2 2 — 230 (His)₆

These proteins were adjuvanted with either Freund's complete adjuvant(FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera weretested against the homologous strain using the bactericidal assay and byELISA.

Results obtained after immunisation with proteins disclosed in refs. 1 &2 were as follows:

Bactericidal titre ELISA n X₁ L₁ X₂ L₂ FCA Alum FCA Alum 2 ORF46.1 — 961(His)₆ 8192 8192 21558 >109350 — 961c (His)₆ 8192  128  9020 76545

Hybrid Proteins—X₁=230

In addition to those disclosed in references 1 & 2, four hybrid proteinswith 230 at the N-terminus were constructed:

# n X₁ L₁ X₂ L₂ 1 2 230 — ORF46.1 (His)₆ 2 2 — 961 (His)₆ 3 2 — 961c(His)₆ 4 2 — 741_(MC58) (His)₆

Hybrid Proteins—X₁=936

In addition to those disclosed in references 1 & 2, seven hybridproteins with 936 at the N-terminus were constructed:

# n X₁ L₁ X₂ L₂ 1 2 936 — ORF46.1 (His)₆ 2 2 — 961 (His)₆ 3 2 —741_(ET37) (His)₆ 4 2 — 741_(MC58) (His)₆ 5 2 — 741_(90/18311) (His)₆ 62 — 741_(95N477) (His)₆ 7 2 — 741 (His)₆

These proteins were adjuvanted with either Freund's complete adjuvant(FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera weretested against various Neisserial strains using the bactericidal assay.Titres using protein #2 were as follows:

Strain^((serogroup)) 2996^((B)) MC58^((B)) 394/98^((B)) 44/76^((B))F6124^((A)) Al hydroxide 16384 32768 1024 2048 <16 FCA 65536 65536 >20488192 2048 (36%)

Titres using protein #4 were as follows:

Strain^((serogroup)) 2996^((B)) MC58^((B)) 394/98^((B)) 44/76^((B))F6124^((A)) Al hydroxide 256 >262144 >2048 32768 8192 FCA1024 >262144 >2048 >32768 >32768

Titres using protein #7 were as follows:

Strain ^((serogroup)) 2996 ^((B)) MC58 ^((B)) 394/98 ^((B)) 44/76 ^((B))F6124 ^((A)) BZ133 ^((C)) Al hydroxide 256 130000 16000 32000 8000 16000

Results obtained after immunisation with proteins disclosed in refs. 1 &2, tested against the homologous strain, were as follows:

Bactericidal titre ELISA n X₁ L₁ X₂ L₂ FCA Alum FCA Alum 2 936 — 741(His)₆ 1024 256 1466 5715 936 >32768 >32768 >109350 >109350

Mixtures of Hybrid Proteins

Mice were immunised with of three proteins adjuvanted with aluminiumhydroxide, either single or in a triple combination: (1) 287_(NZ)-953;(2) 936-741; and (3) 961c. The mixture was able to induce highbactericidal titres against various strains:

2996 ^((B)) MC58 ^((B)) NGH38 394/98 ^((B)) 1144/76 ^((B)) F6124 ^((A))BZ133 ^((C)) C11 ^((C)) (1) 32000 16000 130000 16000 32000 8000 160008000 (2) 256 131000 128 16000 32000 8000 16000 <4 (3) 32000 8000 — — —8000 — 32000 mix 32000 32000 65000 16000 260000 65000 >65000 8000 (X)4000 4000 1000 1000 >4000 1000 4000 n.d. ‘—’ indicates that this straincontains no NadA gene (X) was a combination of protein 287 with outermembrane vesicles, for comparison

Looking at individual mice, the mixture induced high and consistentbactericidal titres:

# 1 2 3 4 5 6 7 8 9 10 2996 32768 16384 65536 32768 32768 65536 6553632768 65536 8192 MC58 65536 32768 65536 65536 65536 8192 65536 3276832768 65536 394/98 65536 4096 16384 4096 8192 4096 32768 16384 819216384

Tandem Proteins

Hybrid proteins of the invention can be represented by formulaNH₂—[—X-L-]-COOH. Where all n instances of -X- are the same basicprotein (either identical, or the same protein from different strains orspecies), the protein is referred to as a ‘tandem’ protein.

Twelve specific tandem proteins are:

# n X₁ L₁ X₂ L₂ 1 2 ΔG741_(MC58) — 741_(MC58) (His)₆ 2 2 ΔG287₂₉₉₆(Gly)₆ ΔG287_(394/98) (His)₆ 3 2 ΔG287₂₉₉₆ (Gly)₆ ΔG287₂₉₉₆ (His)₆ 4 2ΔG287_(394/98) (Gly)₆ ΔG287_(394/98) (His)₆ 5 2 ΔG287_(394/98) (Gly)₆ΔG287₂₉₉₆ (His)₆ 6 2 ΔG287₂₉₉₆ (Gly)₆ ΔG287_(394/98) — 7 2 ΔG287₂₉₉₆(Gly)₆ ΔG287₂₉₉₆ — 8 2 ΔG287_(394/98) (Gly)₆ ΔG287_(394/98) — 9 2ΔG287_(394/98) (Gly)₆ ΔG287₂₉₉₆ — 10 2 ΔG741_(MC58) — 741_(394/98)(His)₆ 11 2 ΔG741_(MC58) — 741_(90/18311) (His)₆ 12 2 ΔG741_(MC58) —741_(95N477) (His)₆

Proteins #1 to #5 have all been expressed in soluble form in E. coli.Expression levels were between 0.24 and 0.50 mg protein per litre ofculture. The tandem proteins were purified and mixed with aluminiumphosphate as an adjuvant. Tandem proteins #2, #4 and #5 adsorbed readilyto aluminium phosphate; adsorption was less complete for tandem proteins#1 and #3.

Allelic Variants—741

Twenty-two polymorphic sequences of 741 were found (SEQ IDs 1 to 22).These and the MC58 sequence are aligned in FIG. 1.

Allelic Variants—NMB1343

Using PCR on 42 strains of meningococcus of various serogroups, the geneencoding NMB1343 protein was found in 24/42 and was absent in 18/42strains (Table 1). The NMB1343 gene was sequenced for 10 of the NMB1343⁺strains (Table 1, column 3). The nucleic acid sequence (and thus aminoacid sequence SEQ ID 23; GenBank AAF41718) was identical in all 10strains.

NMB1343 was also detected in two strains of N. gonorrhoeae (F62 andSN4). The amino acid sequence from gonococcus is SEQ ID 24. An alignmentwith the meningococcal sequence is:

An alignment of the corresponding nucleotide sequences is shown in FIG.2. This shows that the gonococcal sequence has a 4mer insertion in the5′ region of the NMB1343 gene which causes a frameshift and consequentloss of the 5′ methionine residue.

Domain Deletion—961

961 is not present in the N. meningitidis serogroup A genome sequence[81], even though the surrounding regions are conserved (>90%) betweenserogroups A and B. References 11 and 12 disclose polymorphic forms of961. The gene was found to be present in 91% of serogroup B strainsbelonging to hypervirulent lineages ET-5, ET-37 and cluster A4, but wasabsent in all strains of lineage 3 tested. Most of the serogroup Cstrains tested were positive even if not belonging to hypervirulentlineages. The same was true for the serogroup B strains with serotype 2aand 2b. For serogroup A, one strain belonging to subgroup III waspositive whereas the other two strains belonging to subgroup IV-1 werenegative. 961 was absent in N. gonorrhoeae and in commensal species N.lactamica and N. cinerea.

FIGS. 4 and 5 show domains in protein 961.

When the anchor region (domain 9) of protein 961 is deleted (‘961cL’)and expressed in E. coli, the protein is exported in the periplasm andsecreted in the supernatant of the culture.

To investigate this further, deletion mutants in the C-terminal regionof 961 were constructed (961cL-Δaro, 961cLΔcc, 961aL, 961aL-Δ1,961aL-Δ2, 961aL-Δ3) on the basis of structural features (deletions ofaromatic residues in the cases of 961cΔaro mutant, and of coiled-coilregions for the others). These were analysed for expression andsecretion into the periplasm and the supernatant of the culture. In allof these deletion mutants, the protein is produced in large amount, ispresent in periplasmic fraction, and is released in the supernatant ofthe culture.

ΔG287—Cross-Strain Bactericidal Activity

287 was cloned for five different N. meningitidis serogroup B strainsand was manipulated to delete the N-terminus up to the end of thepoly-glycine region and to introduce a C-terminal his-tag. This gavefive ΔG287 proteins. These were adjuvanted with FCA and used to raiseimmune sera in mice, which were then tested for bactericidal activityagainst all five serogroup B strains and also against serogroup A and Cstrains. Bactericidal titres were as follows:

Protein Sera tested for bactericidal activity against strain * strain2996 BZ232 MC58 1000 394/98 F6124 BZ133 2996 16000 128 4096 4096 10248000 16000 BZ232 >8000 256 2048 8000 2048 16000 8000 MC58 >8000 64 >80008000 2048 8000 8000 1000 >8000 64 4096 8000 1024 16000 16000394/98 >16000 128 16000 >2048 >16000 — — * titres against homologousstrain shown in bold

Refolding

To improve the levels of soluble protein for some hybrid proteins,alternative refolding protocols to those disclosed in reference 2 wereadopted.

Inclusion bodies (IBs) were isolated as follows:

-   -   1. Homogenize cells (5 g wet weight) in 25 ml 0.1 M Tris-HCl pH        7, 1 mM EDTA, at 4° C. using an ultraturrax (10 000 rpm)    -   2. Add 1.5 mg lysozyme per gram cells, mix shortly with an        ultraturrax, and incubate at 4° C. for 30 min.    -   3. Use sonication or high-pressure homogenization (French press)        to disrupt the cells.    -   4. To digest DNA, add MgCl₂ to a final concentration of 3 mM and        DNase to a final concentration of 10 μg/ml, and incubate for 30        min at 25° C.    -   5. Add 0.5 vol. 60 mM EDTA, 6% Triton X-100, 1.5M NaCl pH7, to        the solution, and incubate for 30 min at 4° C.    -   6. Spin down inclusion bodies by centrifugation at 31000 g (20        000 rpm) for 10 min, 4° C.    -   7. Resuspend pellet in 40 ml 0.1 M tris-HCl pH 7, 20 mM EDTA,        using an ultraturrax    -   8. Repeat centrifugation step 6.    -   9. The inclusion body pellet may be used, or stored frozen at        −20° C.

Hybrid proteins were expressed in E. coli as follows:

Culture Flask Inclusion volume volume Temp Final body yield Protein(litres) (litres) (° C.) OD₆₀₀ (w/w) ORF46.1-961-His 1 2 37 1.51 33.2%ORF46.1-961c-His 1 2 37 1.6 28.3% 961c-ORF46.1His 1 2 37 1.18 23.5%orf46.1-741 His 5 5 37 12.42 35.2

The pellets were solubilised, refolded, ultrafiltered, dialysed, andprotein was then purified:

ORF46.1-961-His IBs were solubilised as follows: IB proteins wereresuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to afinal protein concentration of 1 mg/ml. To refold the protein, 2 ml ofsolubilised protein was diluted in 400 ml of refolding buffer (0.1M TrisHCl, 1M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 hour at 15°C., resulting in a protein concentration of 5 μg/ml. Subsequently,another 2 ml of the solubilised protein was added and incubated for anadditional hour at the same temperature resulting in a final proteinconcentration of 10 μg/ml. The material was ultrafiltered using a 300 mlAmicon ultrafiltration cell (8400), applying a 3 bar pressure on anAmicon membrane with a 30 kDa cut-off (YM30) resulting in 130 ml finalvolume. The ultrafiltered material was dialysed using a regeneratedcellulose tubular membrane with a 12-14 kDa cutoff (Cellusep—Step bio)for 24 hours against 10 L of 0.1M Tris HCl pH 8.2 buffer. A seconddialysis of 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH8.0 buffer was performed. The dialysed material was centrifuged at 22000rpm for 45 minutes at 4° C. in a Beckman centrifuge rotor JA25.5 Thesupernatant isolated after centrifugation was used for His-tagpurification.

orf 46.1-961c-His IBs were solubilised as follows: IB proteins wereresuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to afinal protein concentration of 1 mg/ml. To refold the protein, 2 ml ofthe solubilised protein was diluted in 400 ml refolding buffer (0.5MTris HCl, 1M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 h at 15°C., resulting in a protein concentration of 5 μg/ml. Subsequentlyanother 2 ml of the solubilised protein was added and incubated for anadditional hour at the same temperature resulting in a final proteinconcentration of 10 μg/ml. The material was ultrafiltered using a 300 mlAmicon ultrafiltration cell (8400), applying a 3 bar pressure on anAmicon membrane with a 30 kDa cut-off (YM30) resulting in 150 ml finalvolume. The ultrafiltered material was dialysed using a regeneratedcellulose tubular membrane with a 12-14 kDa cutoff (Cellusep—Step bio)for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysisof 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0buffer was performed. The dialysed material was centrifuged at 22000 rpmfor 45 minutes at 4° C. in a Beckman centrifuge rotor JA25.5. Thesupernatant isolated after centrifugation was used for His-tagpurification.

961c-orf46.1-His IBs were solubilised as follows: IB proteins wereresuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to afinal protein concentration of 1 mg/ml. To refold the protein, 2 ml ofthe solubilised protein was diluted in 400 ml refolding buffer (0.1MTris HCl, 0.5 M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 h at15° C., resulting in a protein concentration of 5 μg/ml. Subsequentlyanother 2 ml of the solubilized protein was added and incubated for anadditional hour at the same temperature resulting in a final proteinconcentration of 10 μg/ml. The material was ultrafiltered using a 300 mlAmicon ultrafiltration cell (8400), applying a 3 bar pressure on anAmicon membrane with a 30 kDa cut-off (YM30) resulting in 150 ml finalvolume. The ultrafiltered material was dialysed using a regeneratedcellulose tubular membrane with a 12-14 kDa cutoff (Cellusep—Step bio)for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysisof 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0buffer was performed. The dialysed material was centrifuged at 22000 rpmfor 45 minutes at 4° C. in a Beckman centrifuge rotor JA25.5. Thesupernatant isolated after centrifugation was used for His-tagpurification.

orf46.1-741-His IBs were solubilised as follows: IB proteins wereresuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to afinal protein concentration of 10 mg/ml. To refold, 2 ml of thesolubilised protein was diluted in 400 ml of the refolding buffer (0.5MTris HCl, 0.7 M L-arginine, 2 mM EDTA pH 7.2) and incubated for 1 h at15° C., resulting in a protein concentration of 50 μg/ml. Subsequentlyanother 2 ml of the solubilised protein was added and incubated for anadditional hour at the same temperature resulting in a final proteinconcentration of 100 μg/ml. The material was ultrafiltered using a 300ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on anAmicon membrane with a 30 kDa cut-off (YM30) resulting in 120 ml finalvolume. The ultrafiltered material was dialysed using a regeneratedcellulose tubular membrane with a 12-14 kDa cutoff (Cellusep—Step bio)for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysisof 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0buffer was performed. The dialysed material was centrifuged at 22000 rpmfor 45 minutes at 4° C. in a Beckman centrifuge rotor JA25.5 Thesupernatant isolated after centrifugation was used for His-tagpurification.

Compared with proteins purified as described in ref. 2, bactericidalassay titres were as follows:

Reference 2 Refolded Alumin- Alumin- Alumin- ium hy- ium hy- ium phos-Protein CFA droxide droxide MF59 phate ORF46.1-961-His 8192 8192 32768 —— ORF46.1-961c-His 8192 128 <64 8192 — 961c-ORF46.1His 32768 1024 16384— — orf46.1-741 His <4 16 <4  256 —

Similar procedures were used for ORF46.1 to purify the protein from IBswhen expressed with no His-tag (‘ORF46.1K’):

Culture Flask Inclusion volume volume Temp Final body yield Protein(litres) (litres) (° C.) OD₆₀₀ (w/w) orf46.1K 5 5 37 13.7 29.4

IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH8.5 buffer, to a final protein concentration of 10 mg/ml. To refold, 2ml of the solubilised protein was diluted in 400 ml of the refoldingbuffer (0.5M Tris HCl, 0.7 M L-arginine, 2 mM EDTA pH 7.2) and incubatedfor 1 hours at 15° C., resulting in a protein concentration of 50 μg/ml.Subsequently another 2 ml of the solubilised protein was added andincubated for an additional hour at the same temperature resulting in afinal protein concentration of 100 μg/ml. The material was ultrafilteredusing a 300 ml Amicon ultrafiltration cell (8400), applying a 3 barpressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in120 ml final volume. The ultrafiltered material was dialysed using aregenerated cellulose tubular membrane with a 12-14 kDa cutoff(Cellusep—Step bio) for 12 h against 10 L of 50 mM sodium phosphate, 2mM EDTA, pH 7.2 buffer. A second dialysis of 24 h against 10 L of thesame buffer was performed. The dialysed material was centrifuged at22000 rpm for 45 minutes at 4° C. in a Beckman centrifuge rotor JA25.5.The supernatant isolated after centrifugation was used for cationicexchange chromatography. The purification was done on a AKTA explorerchromatography system (Amersham-Pharmacia Biotech) using a 5 ml HiTrapSP sepharose HP column (Amersham-Pharmacia Biotech). The flow rateapplied was of 1.5 ml per minute. The column was washed with 35 ml of 50mM sodium phosphate buffer pH 7.2. A linear gradient (0-1 M NaCl) wasperformed using a 50 mM sodium phosphate buffer pH 7.2. The proteineluted in two peaks at 92 mM and 380 mM NaCl. The fractions constitutingeach peak were pooled and respectively named pool 1 and pool 2.

Compared with proteins purified as described in ref. 2, bactericidalassay titres when adjuvanted with aluminium hydroxide were improved from<4 to 1024. The titre using aluminium phosphate adjuvant with therefolded protein was 2048. ELISA titres were as follows:

Elisa SBA Protein Aluminium adjuvant (M7) (2996) Orf46.1k (pool 1)Hydroxide 3.3 mg/ml 1212 512 Phosphate 0.6 mg/ml 154 1024 Orf46.1k (pool2) Hydroxide 3.3 mg/ml 1085 1024 Phosphate 0.6 mg/ml 250 1024

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 Strain 1343 Sequence Strain classification 72/00 + ET5B:15:P1.7,13,13a 30/00 + ET5 B:15:P1.7,16 39/99 + ET5 C:15:P1.7,1695330 + ET5 B:4:P1.15 M4102 + ET5 nd MC58(21) + + ET5 B:15:P1.7,16bBZ169(7) + + ET5 B:NT:P1.16 BZ83(19) + ET5 B:15:—.— CU385 + + ET5B:4:P1.15 220173I + ET5 NG:4:P1.15 64/96 + + ET5 NG:15:P1.7,16 (carrier)220173I + ET5 B:4:P1.15 (carrier) ISS1071 + nd B:15:P1.7,16 (ET5?)BZ198(2) + + lin.3 B:8:P1.1 980-2543 + + lin.3 B:NT:P1.4 16060 + + otherB:4:P1.14 (carrier) 394-98 + nd B:4:P1.4 (lin 3?) ISS1106 + nd B:4:P1.4(lin.3?) BZ133(10) + + sub I B:NT:—.— S3446 + + nd B:14:P1.23,14ISS1001 + + nd B:14:P1.13 241175I + other NG:21:P1.16 (carrier)171274I + other NG:15:— (carrier) 66/96 + other B:17:P1.15 (carrier)961-5945 − A4 96217 − A4 312294 − A4 90/18311(24) − ET37 93/4286(25) −ET37 M986 − ET37 1000(5) − other NGE28(13) − other carrier NGH38(14) −other carrier BZ232(18) − other F6124(23) − sub III A:—.— C11 − C:— NMB− nd 8047 − nd ISS759 − nd C:2b:P1.2 ISS1113 − nd C:2:P1.5 65/96 − nd4:P1.14 2996(96) − nd B:2b:P1.5,2

REFERENCES The Contents of which are Hereby Incorporated by Reference

-   1—International patent application WO01/64920.-   2—International patent application WO01/64922.-   3—International patent application WO99/24578.-   4—International patent application WO99/36544.-   5—International patent application WO99/57280.-   6—International patent application WO00/22430.-   7—Tettelin et al. (2000) Science 287:1809-1815.-   8—International patent application WO00/66741.-   9—International patent application WO00/71574.-   10—International patent application WO01/04316-   11—International patent application PCT/IB02/03396.-   12—Comanducci et al. (2002) J Exp Med 195:1445-1454.-   13—Vaccine Design: subunit & adjuvant approach (1995) Powell &    Newman (ISBN: 030644867X).-   14—International patent application WO01/52885.-   15—Bjune et al. (1991) Lancet 338(8775): 1093-1096.-   16—Fukasawa et al. (1999) Vaccine 17:2951-2958.-   17—Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.-   18—Costantino et al. (1992) Vaccine 10:691-698.-   19—Costantino et al. (1999) Vaccine 17:1251-1263.-   20—International patent application PCT/IB02/03191.-   21—Watson (2000) Pediatr Infect Dis J 19:331-332.-   22—Rubin (2000) Pediatr Clin North Am 47:269-285, v.-   23—Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.-   24—International patent application WO93/18150.-   25—International patent application WO99/53310.-   26—International patent application WO98/04702.-   27—Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   28—Iwarson (1995) APMIS 103:321-326.-   29—Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   30—Hsu et al. (1999) Clin Liver Dis 3:901-915.-   31—Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.-   32—Rappuoli et al. (1991) TIBTECH 9:232-238.-   33—Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.-   34—Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.-   35—International patent application WO02/02606.-   36—Kalman et al. (1999) Nature Genetics 21:385-389.-   37—Read et al. (2000) Nucleic Acids Res 28:1397-406.-   38—Shirai et al. (2000) J. Infect. Dis. 181 (Suppl 3):S524-S527.-   39—International patent application WO99/27105.-   40—International patent application WO00/27994.-   41—International patent application WO00/37494.-   42—International patent application WO99/28475.-   43—Ross et al. (2001) Vaccine 19:4135-4142.-   44—Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   45—Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.-   46—Dreesen (1997) Vaccine 15 Suppl:S2-6.-   47—MMWR Morb Mortal Wkly Rep 1998 Jan. 16; 47(1):12, 19.-   48—McMichael (2000) Vaccine 19 Suppl 1:S101-107.-   49—Schuchat (1999) Lancet 353(9146):51-6.-   50—WO02/34771.-   51—Dale (1999) Infect Dis Clin North Am 13:227-43, viii.-   52—Ferretti et al. (2001) PNAS USA 98: 4658-4663.-   53—Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages    1218-1219.-   54—Ramsay et al. (2001) Lancet 357(9251):195-196.-   55—Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   56—Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168.-   57—Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.-   58—Goldblatt (1998) J. Med. Microbiol. 47:563-567.-   59—European patent 0 477 508.-   60—U.S. Pat. No. 5,306,492.-   61—International patent application WO98/42721.-   62—Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326,    particularly vol. 10:48-114.-   63—Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or    012342335X.-   64—European patent application 0372501.-   65—European patent application 0378881.-   66—European patent application 0427347.-   67—International patent application WO93/17712.-   68—International patent application WO98/58668.-   69—European patent application 0471177.-   70—International patent application WO00/56360.-   71—International patent application WO00/61761.-   72—Robinson & Tones (1997) Seminars in Immunology 9:271-283.-   73—Donnelly et al. (1997) Annu Rev Immunol 15:617-648.-   74—Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs    9:471-480.-   75—Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447.-   76—Ilan (1999) Curr Opin Mol Ther 1:116-120.-   77—Dubensky et al. (2000) Mol Med 6:723-732.-   78—Robinson & Pertmer (2000) Adv Virus Res 55:1-74.-   79—Donnelly et al. (2000) Am J Respir Crit Care Med 162 (4 Pt    2):5190-193.-   80—Davis (1999) Mt. Sinai J. Med. 66:84-90.-   81—Parkhill et al. (2000) Nature 404:502-506.

1: A protein comprising: (a) the amino acid sequence of SEQ ID NO: 19;or (b) a protein having 70% or greater homology to the amino acidsequence of SEQ ID NO: 19 for use in the prevention and/or treatment ofbacterial meningitis. 2: The protein of claim 1 wherein the protein has80% or greater homology to the amino acid sequence of SEQ ID NO:
 19. 3:A composition comprising the protein of claim 1 or claim 2 and apharmaceutically acceptable carrier. 4: A method of treating a patient,comprising administering to the patient a therapeutically effectiveamount of the composition of claim 3.